Preparation of 3-O-sulfated tetrasaccharide standards was described previously [23 (link)]. Briefly, 400 mg heparin sample was dissolved in 50 mM sodium phosphate buffer (pH 7.4) and was exhaustively digested by heparin lyase 2 (35 IU, activity against heparin). Buffer salts and disaccharides within the product mixture were removed by Bio-Rad P10 column (100 × 50 cm) eluted at 1.2 mL/min with 0.2 M NaCl. The remaining tetrasaccharides mixture was desalted on a Bio-Rad P2 column (100 × 20 cm) and lyophilized. The resulting mixture was fractionated on a semi-preparative strong anion exchange (SAX)-high performance liquid chromatography (HPLC) column Spherisorb S5 (20 × 250 mm Waters, Milford, MA, USA). A gradient of solution A (water, pH 3.5, adjusted with HCl) for 2 min and followed by a linear gradient 0–60% solution B (2.0 M NaCl, pH 3.5, adjusted with HCl) from 2 to 60 min was used at flow rate of 4.0 mL/min with absorbance detected at 232 nm. Individual peaks were desalted on a Bio- Rad P2 column and were further purified by repeated separation on the same SAX-HPLC column. The purified tetrasaccharide standards were quantified by carbazole analysis described previously [31 (link)].
P2 column
Manufactured by Bio-Rad
Sourced in United States
The P2 column is a laboratory equipment used for chromatographic separation and purification of biomolecules. It is designed to efficiently fractionate and isolate target analytes from complex samples, such as proteins, peptides, or nucleic acids.
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4 protocols using p2 column
1
Purification and Quantification of Heparin Tetrasaccharides
2
Site-directed spin labeling and NMR analysis
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Aspartate 31 was converted to Cys in both PEP-19 and PEP(P37G) for site-directed spin labelling with 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (PROXYL; Sigma). A ten-fold molar excess of PROXYL dissolved in ethanol was added to Cys derivatives in 40 mM Tris (pH 8), 2 mM EDTA and 6 M urea, and incubated at room temperature in the dark for 4 h. Unreacted PROXYL was then quenched with 10 mM 2-mercaptoethanol and removed by desalting using a Bio-Rad P2 column. Chemical shifts of amide resonances in 15N-labelled apo C-CaM bound to spin-labelled PEP-19 (PEP-19SL) or PEP(P37G) (PEP(P37G)SL) are almost identical to those observed when bound to PEP-19 or PEP(P37G), which indicates that the introduction of the PROXYL group does not greatly perturb structure. HSQC spectra were collected at 298 K in the absence and presence of 5 molar equivalents of sodium ascorbate to reduce PROXYL to its diamagnetic form. After linear prediction and zero filling, the spectra were fitted using Lorentzian function in each dimension. The PRE effect was measured as a normalized ratio (Iox/Ired) of signal intensities in the oxidized (paramagnetic) and reduced (diamagnetic) state.
3
Quantification and Copper Binding of Ctr1c Peptide
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Apo-Ctr1c was quantified by amino acid analysis by AAA Service Laboratory, Inc. Absorbance was measured from 200 to 360 nm using a Cary300 UV–Vis spectrophotometer by Agilent and the extinction coefficient was calculated. The Ctr1c-peptide was Cu(I)-loaded anaerobically in equimolar ratio with Cu(I)-(CH3CN4)PF6 for 2 h and excess unbound Cu(I) was removed by size-exclusion chromatography using a BioRad P2 column in spectroscopy buffer or by dialysis. ICP-MS results indicated that one Cu(I) ion was coordinated by 3 Ctr1c peptides, similar to what is seen in full-length Ctr1 (De Feo et al. 2009 (link)). Even when copper was added in 3–5 fold excess of peptide, the resulting copper bound remained consistent.
4
Isolation and Fractionation of Human Milk Oligosaccharides
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Fourteen HMO fractions (designated as Fraction 1 -Fraction 14) were produced from pooled HMOs (pHMOs) originally isolated from human milk pooled from over 50 different donors with term infants. First, after centrifugation of the pooled human milk, the lipid layer was removed and proteins were precipitated from the aqueous phase by addition of ice-cold ethanol and subsequent centrifugation. Ethanol was removed from the HMO-containing supernatant by rotoevaporation. Lactose and salts were removed by gel filtration chromatography over a BioRad P2 column (100 cm316 mm, Bio-Rad, Hercules, California, USA) using a semi-automated fast protein liquid chromatography (FPLC) system. Second, pHMOs were separated by charge using
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